The effects of organic waste materials on Miscanthus × giganteus yield and Zn and Ni content

The aim of the experiment was to determine the yield of Miscanthus × giganteus M 19 in the first three years of cultivation and its bioaccumulation of Zn and Ni in aboveground and underground parts in response to different doses of sewage sludge and substrate left after the production of white mushrooms. Miscanthus × giganteus is a grass species that adapts to different environmental conditions and can be grown in various climatic zones of Europe and North America. In April 2018 the experiment was established in a randomized block design and with four replications in central-eastern Poland. Waste organic materials (municipal sewage sludge and mushroom substrate) were applied to the soil in 2018 in the spring before the rhizomes of giant miscanthus were planted. Each year (from 2018 to 2020) biomass was harvested in December. The yield of fresh and dry matter and the total content of Zn and Ni, after wet mineralization of plant samples, were determined by optical emission spectrometry (ICP-OES). After the third year of cultivation, the content of Zn and Ni in rhizomes and in the soil was determined again. In relation to control, an increase in the yield of miscanthus biomass in response to organic waste materials was noted. Plants responded to mushroom substrate (SMS) with the highest average yield (16.89 Mgha−1DM), while on the control plot it was 13.86 Mg ha−1DM. After the third year of cultivation, rhizomes of Miscanthus x giganteus contained higher amounts of Zn (63.3 mg kg−1) and Ni (7.54 mg kg−1) than aboveground parts (40.52 and 2.07 mg kg–1), which indicated that heavy metals were retained in underground parts.


Symbols
Experimental plots Organic waste materials (municipal sewage sludge and mushroom substrate) were applied once in the spring of 2018 before rhizomes were planted.They were mixed with soil to a depth of 25 cm.Municipal sewage sludge was provided by the sewage treatment plant in Siedlce, with its capacity of approx.24 000 m 3 per day.Annually, it produces 1 897 Mg of sewage sludge, an average of 5.2 Mg per day.Mushroom substrate was obtained from a mushroom farm located in the Siedlce district.The producer of the substrate for mushroom cultivation was Unikost, while peat moss for the casing layer was produced by Wokas.The plants took root on all experimental plots.Weeds were controlled mechanically, but not in the third year when hardly any of them were found.The dose of 170 kg ha −1 of N in an organic form was established according to the recommendations of the Nitrates Directive, aimed at reducing water pollution by nitrates from agricultural sources.Pollution prevention measures adopted by the Regulation of the Polish Council of Ministers 19 were also taken into account.

Determination of soil and organic material properties
The following were determined in air-dry soil samples collected from three layers: 0-20 cm, 20-40 cm and 40-60 cm before the experiment started and in those collected only from the arable layer (0-20 cm) in the third year: • granulometric composition using the Bouyoucos-Casagrande hydrometric method modified by Prószyński in accordance with the Polish Standard PN-R-04033 20 and with the classification according to the grain size of soil and of mineral formations 21 , • pH value in H 2 O and in 1 mol l −1 of KCl by the potentiometric method, • total nitrogen (N t ) and carbon (C t ) by elemental analysis using the PerkinElmer® 2400 Series II CHNS/O Elemental Analyzer with a thermal conductivity detector (TCD); • content of total P, K, Zn and Ni (after wet mineralization of soil material with aqua regia) by optical emission spectrometry (ICP-OES) at Eurofins Environment Testing Poland Ltd. in Katowice, formerly the Centre for Environmental Research and Control, • available forms of P and K by the Egner-Riehm method at the Regional Chemical-Agricultural Station in Lublin according to the following Polish Standards: PN-R-04023:1996 and PN-R-04022:1996+Az1:2004, • available forms of Mg by the Schachtschabel method at the District Chemical and Agricultural Station in Lublin according to the PN-R-0420:1994+Az1:2004 Polish Standard.
In organic materials, the following were determined: • dry matter content, after drying the sample at 105 °C until a constant weight; • pH value in H2O and 1 mol l −1 of KCl by the potentiometric method; The uptake of Zn and Ni by Miscanthus × giganteus was calculated based on their total content in aboveground dry matter yield.The bioaccumulation coefficient (BC) of Zn and Ni was calculated as aratio between the average concentration of an element in the plant (C shoot )and in the soil C soil 29 .
BC shoot -coefficient of bioaccumulation of heavy metals in the aboveground parts of giant miscanthus; BC rhizome -coefficient of heavy metal bioaccumulation in giant miscanthus rhizomes.
The translocation factor (TF) of heavy metals (Zn and Ni) was calculated according to the following formula: where: TF-heavy metal translocation factor; C shoot -content of heavy metals in the aboveground parts of miscanthus (mg kg −1 ); C rhizome -content of heavy metals in miscanthus rhizomes (mg kg −1 ); C soil -content of heavy metals in the soil (mg kg −1 ).
The Pearson linear correlation coefficient between the following variables (arranged in pairs) was calculated: total Zn and Ni content in the soil, in the aboveground parts of Miscanthus × giganteus, in its rhizomes, Zn and Ni content in biomass, bioaccumulation coefficient and translocation factor.

Results and discussion
What determines the value of organic waste materials, apart from their nutrient content and yield-increasing potential, is the dynamics of their decomposition in soil 6 .As regards municipal sewage sludge, its excessive content of heavy metals and soil microbiological contamination may exclude its application to crops.However, when its composition indicates that it can be used in agriculture, it has manifold benefits even though its use is still at a low level.This is due to insufficient research on its properties and on its effects on soil, plants and the environment, but also because of reluctant social attitude.Municipal sewage sludge used in the present experiment contained high amounts of dry matter (93%),but also of N, with 40.50 g kg −1 DM, and of P, with 19.81 g kg −1 DM (Table 2).According to the literature 30 , its K amounts are low, which was also confirmed by the present experiment (2.56 g kg −1 ).Even though in the present research pH of municipal sewage sludge was slightly acidic (pH 6.4), its application to acidic and degraded soils does not contribute to their acidification.In Poland, according to the Regulation of the Ministry of Climate and the Environment of 2020 31 , the pH value of soil treated with municipal sewage sludge cannot be lower than 5.6.2), with Jordan et al. 32 reporting similar amounts.N concentration of 20.9 g kg −1 was also high, while organic C concentration, as an indicator of soil biological activity, was 284 g kg −1 .The C:N ratio was 13.59, indicating considerable mineralization of mushroom substrate N compounds with release of its nutrients.The concentration of mushroom substrate P was 8.86 g kg −1 , with 11.21 g kg −1 of K.In the organic waste materials, the content of Zn and Ni did not exceed the standards set by the Regulations of the Ministry of Climate and the Environment 24 , which meant that their application to Miscanthus × giganteus was allowed.
The fresh matter yield of giant miscanthus aboveground parts, as an average of treatment combinations and years of research, was 26.22 Mg ha −1 (Table 3).It varied significantly over growing periods and was the lowest in the first one.In subsequent years, the yields were higher.The low value in the first year might have been caused by the insufficient development of roots, necessary for plant growth 33,34 .The highest yield of 39.58 Mg ha −1 FM, average across treatment combinations, was recorded in the third year.The biomass yield of giant miscanthus harvested in the third growing period was higher by 160% than in the first and by 60% in the second.Gubiśová et al. 35 report that miscanthus reaches its full productivity in the third year after its plantation.
The yield of Miscanthus × giganteus fresh matter significantly varied across treatment combinations and was considerably higher on fertilized plots than on the control one.In the first year the highest fresh matter yield of 17.50 Mg ha −1 was recorded on the plot where the lowest dose of sewage sludge in combination with the highest dose of mushroom substrate was applied (SS 25 + SMS 75 ).The next highest value of 17.10 Mg ha −1 was noted on the plot with mushroom substrate applied on its own (SMS), while the lowest yield was on the control plot (12.50 Mg ha −1 ).A similar tendency was observed in the second year, with the highest fresh matter yield in response to the application of mushroom substrate on its own and in combination with sewage sludge, with 28.50 and 25.95 Mg ha −1 , respectively.In the third year, the highest value was recorded on the plot with municipal sewage sludge (43.10 Mg ha −1 ) and the lowest on the control plot (36.00 Mg ha −1 ).
Thus, Miscanthus × giganteus dry and fresh matter yields varied across treatment combinations, which can be explained by the fact that the plants on different plots varied in their proportion of leaves in relation to shoots.The share of leaves can be up to 38% on average, and their water absorption capacity is higher 34 .
In the third year, the average dry matter yield of Miscanthus × giganteus, with 21.58 Mg ha −1 ,was more than twice as high as in the first year.It significantly varied throughout the experiment due to a growing number of shoots and because plants were taller year by year, with Angelini et al. 36 having observed the same.Compared to control, the organic materials used in the experiment had a significant effect on the average dry matter yield of giant miscanthus, except for the plot on which sewage sludge and mushroom substrate were applied together in equal doses (SS 50 + SMS 50 ), both containing the same amount of nitrogen.On average, for three years, plants responded to mushroom substrate (SMS)with the highest fresh and dry matter yields.
Clifton-Brown and Lewandowski 33 and Keymer and Kent 37 found that giant miscanthus yield was affected by temperature distribution during the growing period and by soil moisture, but to a smaller extent by fertilizer treatment, especially by nitrogen supply.In the present experiment optimal thermal and moisture conditions were in June, July and October 2018 (Fig. 1).In other months, weather conditions varied.In June 2019 and 2020 (the second and third years) extreme droughts were recorded.Extremely wet conditions were in May and June 2020.The most unfavourable conditions for giant miscanthus were in 2019, as they ranged from extremely dry to moderately dry in each month with the exception of May (Fig. 1).
Zn concentration in plant biomass, average across treatment combinations, was the highest (52.90 mg kg −1 DM) in the first year (Table 4).In the third year of the experiment, it was twice as low (28.45 mg kg −1 DM), because biomass yield was more than twice higher than in the first (Table 3).Bilandžija et al. 38 report a slightly higher Zn concentration in the biomass of the same plant harvested in winter (55.2 mg kg −1 DM).On the other hand, According to Bosiacki's 40 studies on phytoextraction potential, Miscanthus × giganteus is not a Zn and Cu hyper accumulator.The literature has confirmed its tolerance to increased concentrations of heavy metals in soil 41 .According to the above authors, osier willow (Salix viminalis L.) has a much greater potential to accumulate heavy metals than giant miscanthus.In the present experiment the highest concentration of Zn was found in plants treated with mushroom substrate (SMS) in the first and second years of cultivation, with 81.30 and 51.31 mg kg −1 DM, respectively.On the other hand, its lowest amount (35.34 mg kg −1 DM), as an average of all growing periods, was in plants treated with the highest dose of sewage sludge together with the lowest dose of mushroom substrate (SS 75 + SMS 25 ).
Across treatment combinations, the highest average Ni concentration (2.64 mg .kg -1 DM) was in the aboveground parts of giant miscanthus in the first year (Table 5).Its decrease was noted in subsequent years of the experiment, due to its dilution with biomass yield that increased with the age of the plants (Table 3).The average concentration of this metal in plants across growing periods was 2.07 mg kg −1 DM.Compared to the results reported by Kotecki 34 and Bosiacki et al. 39 , the content of Ni in the biomass of giant miscanthus was much lower.According to Bosiacki et al. 39 ,miscanthus Ni concentration ranged from 5.29 to 5.73 mg kg −1 DM,depending on the growing period.The highest bioaccumulation of Ni (an average from the years of research) was in response to the combinations of municipal sludge with mushroom substrate: SS 25 + SMS 75 and SS 75 + SMS 25 , with 2.77 and 2.27 mgkg −1 DM, respectively.On the other hand, the lowest value was recorded on the control plot (1.77 mg kg −1 DM).On the remaining experimental units, the concentration of Ni in plants did not exceed 2 mg kg −1 DM.Bosiacki et al. 39 report higher amounts of Ni in control plants than in those treated with mineral nitrogen.Kalembasa and Malinowska 42 observed that the harvest date affected the content of Ni in the aboveground parts of Miscanthus sacchariflorus.Additionally, plants harvested in summer contained more Ni than those harvested in winter.This chemical element is highly mobile in plants, and its excess can cause disorders of basic physiological processes such as photosynthesis and transpiration 29 .
Extraction of Zn from Miscanthus × giganteus biomass significantly varied across treatment combinations and years of research (Table 6).On average, the plants absorbed the most Zn, 61% and 36.5% more than on the control plot, in response to mushroom substrate (SMS) and municipal sewage sludge (SS) applied separately.Among all treatment combinations significantly the lowest Zn content was noted in giant miscanthus growing on the control plot.There were no significant differences in Zn accumulation between plants treated with SS 75 + SMS 25 and SS 50 + SMS 50 (sewage sludge and mushroom substrate combinations).The average extraction of Zn from plants growing on those plots did not differ much.The amounts of this metal accumulated by plants in subsequent years increased with yield and were the highest in the third one despite the lowest Zn content (Table 4).According to Kabata-Pendias and Pendias 29 , cereals and spinach (Spinacia oleraceae) are the most sensitive plants to Zn content in soil, and the greatest risk of toxicity is on sandy acidic soils.This was also confirmed by the reports of McBride 43 .Extraction of Ni was slightly different from that of Zn (Table 7).The highest value was noted in plants treated with SS 25 + SMS 75 and SS 75 + SMS 25 ,71% and 45% higher than on the control plot, where the lowest amounts were noted.On the plots with SS and SS 50 + SMS 50 the content of Ni in Miscanthus × giganteus yield did not differ statistically.The uptake of this metal with biomass significantly varied over growing periods, with the highest value in the third year and the lowest in the first.Jakubus 44 found that nutrients in organic waste materials had a limited impact on the chemical composition of energy crops.According to Kalembasa and Malinowska 45 , due to the residual effect the intensity of metal uptake by Miscanthus sacchariflorus biomass increased in the third and partly in the fourth year after sewage sludge application.The authors report that sewage sludge application resulted in a threefold increase in Ni uptake, a twofold increase in Cr and Cu uptake and in a greater accumulation of Zn and Cd in the third year.Deans et al. 46 found that heavy metal uptake was dependent on miscanthus maturity and was proportional to the soil content of heavy metals.According to some studies on dicotyledonous plants, the bioaccumulation of Zn by plants varies depending on its availability in soil and on growing conditions [47][48][49][50] .
The content of Zn and Ni in Miscanthus × giganteus rhizomes after 3 years significantly varied depending on the residual effect of the waste materials (Table 8).It was the highest in response to the greatest dose of sewage sludge applied together with the smallest amount of mushroom substrate (SS 75 + SMS 25 ).Compared to other fertilized plots, the content of Zn in rhizomes was significantly lower after the application of SMS, probably because some amounts had been translocated to aboveground parts.According to Table 6, the amounts of this element in rhizomes from the SS 75 + SMS 25 plot was the highest of all treatment combinations.On the control plot, Zn content in miscanthus rhizomes was higher than on plots with SMS or with SS, by 38% and 15.4%, respectively (Table 8).
The lowest content of Ni was in rhizomes from the control plot, almost two times lower than on plots with SS 75 + SMS 25 , SS 25 + SMS 75 and SS.After the third year the average amounts of Ni in rhizomes, with 7.54 mg kg −1 DM, were over three times higher than in aboveground parts, with 2.07 mg kg −1 DM (Table 5).Rhizomes, on average, also accumulated more Zn (63.3 mg kg −1 DM) than aboveground parts (40.52 mg kg −1 DM), but the difference was not as great as for Ni.In the third year a much higher bioaccumulation coefficient (BC) of the heavy metals, especially of Ni, was found in giant miscanthus rhizomes than in aboveground parts (Table 8).The average BC value of Ni in rhizomes was 0.887whereas in aboveground parts it was 0.189.In the case of Zn it was 0.232 in rhizomes and 0.104 in shoots and leaves.The above values meant that waste materials used in the experiment did not pose a threat to the environment.On this basis, it can be assumed that the root system was a serious barrier to the movement of these elements to aboveground parts.The accumulation of metals in rhizomes and aboveground parts was dependent primarily on the type of metal and, to a lesser extent, on the residual effect of waste materials.This was confirmed by Kloke et al. 51 , who reported that bioaccumulation coefficients in the soil-plant system for Cd and Zn ranged from 1 to 10, while for Pb from 0.01 to 0.1.The translocation factor (TF) is used to determine heavy metal movement from plant roots to aboveground parts.Based on heavy metal concentration in the plant, its phytoextraction capacity can be estimated.According to statistical calculations, the residual effect of the organic waste materials had a significant impact on the translocation factor in the rhizome-aboveground system of giant miscanthus in the third year (Table 8).The Ni translocation factor was significantly the highest on the control plot (0.333).In the case of Zn, the highest TF value (0.630) was recorded on plots with sewage sludge (SS).Mobility of heavy metals in soil and plants and their bioaccumulation depend primarily on soil and environmental conditions, plant species and the type of metal 52 .Ociepa et al. 48noted a much higher translocation of Zn from prairie cordgrass (Spartina pectinata) roots to aboveground parts than in the case of Pb.Their accumulation in individual parts of the grass was dependent mainly on the type of metal.
Compared to the first year, at the end of the third one increased content of Zn and Ni was noted in the soil to which waste materials were applied (Figs. 2 and 3, Table 1).In soil from the control plot, their content was significantly the lowest.The highest amounts of Zn were recorded in soil treated with municipal sewage sludge (SS), and in the case of Ni with sewage sludge applied together with mushroom substrate (SS 50 + SMS 50 ).Malinowska 53 observed that soil Zn concentration increased even twice after the application of sewage sludge, and the increase was dependent on the dose.After the third year of miscanthus cultivation, soil content of both heavy metals was significantly higher on plots with sewage sludge (SS) than on those with mushroom substrate (SMS): by 20.3% in the case of Zn and by 17.2% in the case of Ni.This was due to the difference in the chemical composition of the organic wastes.In Poland, according to the Regulation of the Ministry of the Environment 54 , the content of Zn and Ni in agricultural soils designated as subgroup II-2 (i.e.light mineral soils with pH above 6.5) should not exceed 500 mg Zn kg −1 and 150 mg Nikg −1 .In the present experiment their soil content after the third year of Miscanthus x giganteus cultivation was much lower.
A statistically significant negative correlation (r = − 0.875) was noted between Zn content in soil and BC of giant miscanthus aboveground parts (Table 9).In turn, between Zn content in soil and BC of rhizomes a significant positive correlation of r = 0.843 was noted.As regards the relationship between the Zn translocation factor (from rhizomes to aboveground parts of giant miscanthus) and Zn content in the soil, a weak positive correlation www.nature.com/scientificreports/ was found, not statistically significant.The content of Ni in rhizomes was significantly positively correlated with its content in the soil, with r = 0.985 (Table 10).A significant negative relationship was found between the translocation factor of Ni (from rhizomes to aboveground parts)and its content in soil, with r = − 0.832.Finally, it should be emphasized that bioaccumulation of chemical elements in the soil-plant system and their movement to individual parts of plants depend primarily on soil conditions and on the metal.For each heavy metal the correlation between its content in the soil and in the plant was different and so were BC and TF coefficients.On the basis of the results it should be concluded that Miscanthus × giganteus rhizomes constitute a barrier that stops Zn and Ni movement, which is an extremely positive observation, making miscanthus biomass a very useful energy resource.

Conclusions
The yield of Miscanthus × giganteus fresh and dry matter significantly varied across treatment combinations and growing seasons.The highest average dry matter yield was noted in response to the application of mushroom substrate either on its own(SMS)or in combination with municipal sewage sludge (SS 75 + SMS 25 ) and the lowest on the control plot.In the third year of cultivation dry matter yield was the highest with 21.58 Mg kg −1 .As an  average of all treatment combinations, the highest but still moderate content of Zn and Ni in the aboveground parts of giant miscanthus was in the first year of the study.The uptake of the heavy metals by Miscanthus × giganteus significantly varied across treatment combinations and growing periods.The highest accumulation of Zn was recorded in plants treated with SMS and SS, and of Ni on plots with SS 25 + SMS 75 and SS 75 + SMS 25 .At the end of the third growing period, giant miscanthus rhizomes contained much higher amounts of Zn and Ni than aboveground parts.Bioaccumulation of heavy metals in energy crops may contribute to the emergence of new environmental problems.Because of residues from combustion processes of energy crops with considerable quantities of heavy metals, plants grown on soil treated with organic waste may be classified as hazardous for the environment.However, despite soil treatment with municipal sewage sludge, in the present experiment biomass did not pose a threat in terms of its content of selected heavy metals.Compared to the first year, in the third one, an increased soil content of Zn and Ni was found, significantly higher on the plot with municipal sewage sludge (SS) than on that with mushroom substrate (SMS).Sewage sludge can contaminate the soil and cause excessive accumulation of heavy metals in the plant.Therefore, it is necessary to strictly comply with the legal regulations limiting its use and to select such plants as Miscanthus × giganteus, accumulating heavy metals in the underground part.

Table 1 .
Soil chemical properties before the experiment.

Table 2 .
Chemical properties and dry matter content of organic waste materials.

waste material pH H2O pH KCl DM C org C:N N P K Zn Ni (%) (g kg −1 ) (g kg −1 ) (mg kg −1 )
Mushroom substrate used in the experiment contained as much as 30% of dry matter (Table

Experimental plots (A) Control plot SS SS 75 + SMS 25 SS 50 + SMS 50 SS 25 + SMS 75 SMS Mean
34cording to Bosiacki et al.39, it ranged from 39.04 to 41.97 mg kg −1 DM depending on the growing season.Lower Zn content was noted by Kotecki34in giant miscanthus harvested in October.

Table 4 .
Concentration of Zn in Miscanthus × giganteus biomass (mgkg −1 DM).Within a row, different uppercase letters indicate a significant difference, within a column, different lowercase letters indicate a significant difference.SS-municipal sewage sludge dose introducing 170 kg N ha −1 ; SMS-mushroom substrate dose introducing 170 kg N ha −1 ; SS 75 + SMS 25 ; SS 50 + SMS 50 ; SS 25 + SMS 75 -municipal sewage sludge used together with mushroom substrate in various proportions, each dose introducing 170 kg N ha −1 .

Table 5 .
Concentration of Ni in Miscanthus × giganteus biomass (mg .kg -1 DM).Within a row, different uppercase letters indicate a significant difference, within a column, different lowercase letters indicate a significant difference.SS-municipal sewage sludge dose introducing 170 kg N ha −1 ; SMS-mushroom substrate dose introducing 170 kg N ha −1 ; SS 75 + SMS 25 ; SS 50 + SMS 50 ; SS 25 + SMS 75 -municipal sewage sludge used together with mushroom substrate in various proportions, each dose introducing 170 kg N ha −1 .

Table 8 .
Zn and Ni content in rhizomes, bioaccumulation coefficient and translocation factor in the third year of Miscanthus × giganteus cultivation.Within a column, different lowercase letters indicate a significant difference.SS-municipal sewage sludge dose introducing 170 kg Nha −1 ; SMS-mushroom substrate dose introducing 170 kg Nha −1 ; SS 75 + SMS 25 ; SS 50 + SMS 50 ; SS 25 + SMS 75 -municipal sewage sludge used together with mushroom substrate in various proportions, each dose introducing 170 kg Nha −1 .

Table 9 .
Linear correlation coefficient between Zn content in soil, in aboveground parts, in rhizomes, uptake with biomass yield, bioaccumulation coefficient and translocation factor (all arranged in pairs).*Significant relationship p ≤ 0.05.

Table 10 .
Linear correlation coefficient between Ni content in soil, in aboveground parts, in rhizomes, uptake with biomass yield, bioaccumulation coefficient and translocation factor (all arranged in pairs).*Significant relationship p ≤ 0.05.